Cloning, sequencing, expression, and purification of aspartic proteases isolated from two human Demodex species.

Aspartic proteases (ASPs) are important hydrolases for parasitic invasion of host tissues or cells. This was the first study on Demodex ASP. First, the complete coding sequence (CDS) was amplified, cloned and sequenced. Then, the protein physical and chemical properties was analysed. Finally, the recombinant plasmid, expression and purification system was established. Results showed that the lengths of CDS of Demodex folliculorum and D. brevis were 1161 and 1173 bp, respectively. The molecular weight of the protein was approximately 40 KDa. It contained an aspartic acid residue, a substrate-binding site and signal peptide, yet lacked a transmembrane domain and was located in the membrane or extracellular matrix. The phylogenetic and conserved motif analyses showed that D. folliculorum and D. brevis clustered separately and then formed a single branch, which finally clustered with other Acariformes species. The prokaryotic expression systems for recombinant ASP with His-tag (rASP-His) and GST-tag (rASP-GST) were constructed. The inclusion bodies of rASP-His were renaturated by gradient urea and purified using NI beads, while those of rASP-GST were renaturated by sarkosyl and Triton X-100 and purified using GST beads. Conclusively, the prokaryotic expression and purification system of Demodex rASP was successfully established for further pathogenic mechanism research.

Classification and identification of mosquitoes in China based on rDNA 28S D5 region.

Accurate classification and identification of mosquitoes are essential for the prevention and control of mosquito-borne diseases. In this study, adult mosquitoes were collected from 15 cities across 14 provinces in China. They were identified morphologically with the dominant species determined. Furthermore, representative samples were identified at the molecular level based on rDNA 28S D5. In total, 880 adult mosquitoes were collected belonging to Culex (266), Aedes (473), Armigeres (13), and Anopheles (5). Aedes albopictus and "C. pipiens subgroup" were the dominant species. A total of 140 sequences of 28S D5 region (68 for "C. pipiens subgroup", 51 for Ae. albopictus, 18 for Ar. subalbatus, and three for An. sinensis) ranging from 148 to 161 bp were obtained, with 100 % success of amplification and sequencing. Molecular identification were consistent with morphological classification. Sequence analysis showed that "C. pipiens subgroup" was identified into three clades: the traditional C. pipiens subgroup (Clade I), the newly discovered C. cf. perexiguus (Clade II), and C. new sp. (Clade III). Clade I contained the most abundant haplotypes (16) widely distributed without geographical differences. Clade II included six haplotypes that were aggregately distributed south of the Yangtze River. Only three sequences in Clade III showed two haplotypes with no geographical differences. Further morphological comparisons demonstrated differences in body color, beaks, and abdomens among the three clades. In conclusion, the rDNA 28S D5 region could effectively distinguish Culex, Aedes, Armigeres, and Anopheles species at the lower category level, demonstrating its potential as a mini-DNA barcode for mosquito identification.

Exploration of Multi-Gene DNA Barcode Markers to Reveal the Broad Genetic Diversity of Field Ticks (Acari: Ixodidae) in a Tropical Environment of Hainan Island, China.

Ticks are hematophagous arthropods and obligate ectoparasites of humans and other animals. This study focused on the molecular discrimination of ticks in the tropical environment of Hainan according to multi-gene DNA barcode markers with the expectation of accurately distinguishing species. A total of 420 ticks, including 49 adult ticks, 203 nymphal ticks, and 168 larval ticks, were collected in the field, and the 49 adult ticks were identified as Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis longicornis. The mitochondrial 16S rRNA, ribosomal 28S rRNA D2, and ribosomal internal transcribed spacer 2 (ITS2) regions were used as DNA barcode markers to discriminate species. According to basic local alignment search tool analysis against the GenBank database, 16S rRNA positively identified ticks in the Rhipicephalus, Dermacentor, and Haemaphysalis genera; the 28S rRNA D2 region identified ticks in the Rhipicephalus and Dermacentor genera; and ITS2 identified ticks as D. marginatus. Pairwise sequence comparisons based on these three regions were visualized with a Sequence Demarcation Tool matrix. Substitution saturation tests using data analysis and molecular biology and evolution revealed little substitution saturation (Iss < Iss.c, p < 0.05) in the 16S rRNA region for the Haemaphysalis genus; 28S rRNA D2 region for the Rhipicephalus, Dermacentor, and Haemaphysalis genera; and ITS2 region for the Rhipicephalus and Dermacentor genera. Distinctive sequences for which it is difficult to obtain good matches with the sequences available in GenBank exist in the ticks of Hainan. Future studies should obtain complementary sequences to refine and update the database for the molecular characterization of ticks.

Secondary structure construction and molecular identification of rRNA 18S V4 region E23-5-E23-6 of parasitic lice of Hominidae.

The parasitic lice of Hominidae are a class of blood-sucking insects, having a large fragment expansion region in ribosome 18S V4 region. In this study, the value of the E23-5-E23-6 stem-loop structure in the insertion region for molecular identification of lice were explored through motif analysis and secondary structure construction. Five pubic lice samples from China were morphologically identified, and primers for the rRNA 18S V4 region were designed for molecular identification. The V4 sequence of the parasitic lice of Hominidae was retrieved from GenBank for sequence analysis. The five samples were identified as pubic lice based on V4 region, which were of the same specie but geographically different from Australian strains in Genbank, with the identity of 99.06-99.46%. Compared with the human lice, both the chimpanzee lice and pubic lice had large indel fragments in the V4 region. Comparison results showed that Muscle and MAFFT had better alignment and phylogeny results than Clustal. The large expansion region, comprising E23-5 and E23-6, was located between E23-4 and E23-7. The V4 secondary structure showed that the stem-loop structures of the lice parasitizing on pubic area, human, and chimpanzee were different in the E23-5 and E23-6, which could effectively distinguish the three parasitic lice and divide the human lice into five genotypes. This is suitable not only for the identification of three lice species in higher taxonomic ranks but also for genotype identification of human lice in lower taxonomic ranks. The difference between the stem-loop structure is more intuitive than that between the primary sequences.

De novo transcriptome sequencing and functional annotation of Demodex canis.

Demodex canis is an important parasitic mite causing skin lesions in dogs. However, molecular studies on this species are limited because of the lack of transcriptomic data. To obtain functional genes of D. canis, mites were collected and RNA was extracted for transcriptome sequencing and functional annotation. Coding sequences (CDSs) of important functional genes were screened and identified using bioinformatics strategies. Six complete CDSs were amplified by specific primers, cloned and sequenced. Results demonstrated that the quantity of the RNA extracted from 100 mites was 12.8 ng and the RNA integrity number was 5.4, indicating that it could be used to construct a cDNA library. Transcriptome sequencing yielded 263,619 unigenes, of which 151,048 (57.3%) were functionally annotated. Using bioinformatics strategies, 58 total CDSs were identified as homologous to those of closely related mite species, including 16 types of allergen genes, 13 types of protease genes, and nine types of motion-related genes. The verified CDSs were almost identical to the CDSs of unigenes. Phylogenetic analyses of tropomyosin further revealed that the verified CDSs clustered successively with those of Demodex species and Tetranychus species in Raphignathae, which agreed with the morphological classification, demonstrating the reliability of the transcriptomic data. In conclusion, this study is the first to successfully sequence transcriptome of D. canis, perform functional annotation, and verify CDSs, which will provide ample data for further studies on the functional genes and molecular pathogenic mechanism of D. canis.

Morphological and Molecular Identification of Oestrus ovis (Diptera: Oestridae) Larvae Collected from a Chinese Patient with Conjunctival Myiasis.

PURPOSE: Human conjunctival myiasis, which is often misdiagnosed or missed clinically, is commonly caused by Oestrus ovis larvae. Here, pathogenic identification was performed for two maggots collected from a patient from China, to provide a clinical scientific basis for diagnosis and treatment. METHODS: Morphological identification was performed using a microscope. Oestrus mtDNA cox1 and rDNA 28S were selected as target genes for duplex PCR amplification, followed by cloning, sequencing, and identification. RESULTS: Morphological examination showed that the maggots were approximately 1.0-1.5 mm long, long-oval-shaped, segmented, and covered with small spines, with a pair of hooks in the scolex and claw-like spines at the telson. Therefore, they were identified as the first-instar larvae of O. ovis. Duplex PCR detected products of approximately 400 and 200 bp, consistent with the size of designed cox1 and 28S D7a gene fragments, respectively. Sequences of cox1 and 28S D7a from the samples in question had 99.5-100.0% and 96.2-100.0% similarity (respectively) to GenBank sequences of O. ovis specimens known to parasitize sheep, goats, and humans. However, some 28S D7a sequences exhibited 89.7-90.6% similarity to GenBank sequences of Oestrus sp. known to parasitize Capra pyrenaica (Artiodactyla: Bovidae) (Iberian ibex). Therefore, we considered that the larvae infecting the patient originated from sheep or goats, not Iberian ibex. The phylogenetic trees supported this conclusion. CONCLUSION: This study implemented the first duplex PCR molecular identification of O. ovis larvae parasitizing human eyes in China as a complementary approach to morphological identification. Our results indicate that molecular tools can be utilized to aid in the diagnosis of opthalmomyiasis.

Establishment of purification method for prokaryotic expression of Serpin gene for Dermatophagoides farinae.

This study aimed to develop an effective method for the expression and purification of the Dermatophagoides farinae serpin protein and to establish an experimental foundation for elucidating its role in the temperature stress response. The total RNA of D. farinae was extracted, and specific primers were designed for serpin amplification. Serpin was joined with pET32a vector and transformed into BL21 (DE3) cells. Expression of recombinant proteins was induced. Proteins were extracted by enzymatic lysis or enzymatic lysis combined with ultrasonication. Recombinant proteins were purified by Ni-NTA method. SDS-PAGE was conducted to evaluate protein expression, extraction, and purification efficiency. Agarose gel electrophoresis and sequencing analysis showed that the amplified serpin open reading frame was 1284 bp, encoding a hydrophilic and stable protein with a relative molecular weight of 48.30 kD. SDS-PAGE demonstrated that there was a specific band at 55-70 kD, which was consistent with the predicted size of the recombinant pET32a-Serpin protein. Enzymatic lysis combined with 30% ultrasonic power promoted the release of soluble protein more effectively than enzymatic lysis alone. 16 °C for 4 h was optimal for inducing expression. The optimal imidazole concentrations for washing non-His-tagged protein and eluting His-tagged protein were determined to be 20 mM and 200 mM, respectively. In this study, A prokaryotic expression and purification system for the D. farinae serpin protein was successfully established, providing a technical reference for functional gene research in mites at the protein level.

Molecular identification and DNA barcode screening of acaroid mites in ground flour dust.

Molecular identification of acaroid mites is difficult because of the scarcity of molecular data in GenBank. Here, acaroid mites collected from ground flour dust in Xi'an, China, were preliminarily morphologically classified/grouped. Universal primers were then designed to amplify and screen suitable DNA barcodes for identifying these mites. Sixty mite samples were morphologically classified into six groups. Groups 1-2 were identified to Dermatophagoides farinae and Tyrophagus putrescentiae, while Groups 3-6 were not identified to the species level. ITS2 exhibited higher efficiency in molecular identification in comparison with COI, 12S, and 16S. Groups 1-6 were identified as D. farinae, T. putrescentiae, Suidasia nesbitti, Chortoglyphus arcuatus, Lepidoglyphus destructor, and Gohieria sp., respectively. The phylogenetic results were consistent with the morphological classification. Group 6 was further identified as G. fusca according to the morphology of the reproductive foramen. We conclude that the use of ITS2 and the availability of universal primers provide an ideal DNA barcode for molecular identification of acaroid mites. The use of multiple target genetic markers in conjunction with morphological approaches will improve the accuracy of Acaridida identification.

Molecular Identification, Transcriptome Sequencing and Functional Annotation of Pulex irritans.

PURPOSE: Pulex irritans are vectors of various zoonotic pathogens. However, molecular studies on P. irritans and flea-borne diseases are limited due to the lack of molecular data. This study aimed to conduct transcriptome sequencing, functional annotation, and pathogen analysis of P. irritans. METHODS: Fleas collected from a dog were identified morphologically and molecularly. RNA was extracted for transcriptome sequencing and functional annotation. Open reading frames (ORFs) of unigenes were confirmed by employing bioinformatics strategies, and maximum likelihood (ML) trees were reconstructed based on the highly expressed genes of ejaculation globulin-specific 3-like protein, salivary protein, and actin for phylogenetic relationship analysis. RESULTS: The obtained mitochondrial 16S rRNA gene sequences showed 99.71% of similarity with P. irritans obtained from GenBank database. Transcriptome sequencing generated 74,412 unigenes, of which 53,211 were functionally annotated. A total of 195 unigenes were assigned to fleas, of which 69 contained complete ORFs. Phylogenetic trees of both ejaculatory globulin and salivary protein genes demonstrated that P. irritans first clustered with Pulicidae sp., indicating the reliability of transcriptome data. It is noteworthy that 1070 unigenes were assigned to Hymenolepis microstoma and Dipylidium caninum, of which 62 contained complete ORFs. The phylogenetic tree of the actin gene showed that the unigenes had closer relationships with Echinococcus sp., suggesting the role of P. irritans as intermediate hosts of tapeworms. CONCLUSION: The results of this study provide the possibility for functional exploration of important genes and lay foundations for the prevention and control of P. irritans and flea-borne diseases.

Temperature stress response: A novel important function of Dermatophagoides farinae allergens.

Dermatophagoides farinae, an important pathogen, has multiple allergens. However, their expression under physiological conditions are not understood. Our previous RNA-seq showed that allergens of D. farinae were up-regulated under temperature stress, implying that they may be involved in stress response. Here, we performed a comprehensive study. qRT-PCR detection indicated that 26 of the 34 allergens showed differential expression. Der f1 had the most abundant basic expression quantity. Der f 28.0201 (HSP70) and Der f3 had the same regulation pattern in 9 highly expressed transcripts, which only up-regulated at 41 °C and 43 °C, but Der f 28.0201 showed stronger regulation than Der f 3 (19.88-fold vs 6.02-fold). Whereas Der f 1, 2, 7, 21, 22, 27, and 30 were up-regulated under both heat and cold stress, and Der f 27 showed the strongest regulation ability among them. Der f 27 showed more significant up-regulation than Der f 28.0201 under heat stress (23.59-fold vs 19.88-fold), and Der f27 had more obvious up-regulation under cold than heat stress (30.70-fold vs 23.59-fold). The expression of Der f 27, 28.0201 and 1, and D. farinae survival rates significantly decreased following RNAi, indicating the upregulation of these allergens under temperature stress conferred thermo-tolerance or cold-tolerance to D. farinae. In this study, we described for the first time that these allergens have temperature-stress response functions. This new scientific discovery has important clinical value for revealing the more frequent and serious allergic diseases caused by D. farinae during the change of seasons.

Divergent domains of 28S ribosomal RNA gene: DNA barcodes for molecular classification and identification of mites.

BACKGROUND: The morphological and molecular identification of mites is challenging due to the large number of species, the microscopic size of the organisms, diverse phenotypes of the same species, similar morphology of different species and a shortage of molecular data. METHODS: Nine medically important mite species belonging to six families, i.e. Demodex folliculorum, D. brevis, D. canis, D. caprae, Sarcoptes scabiei canis, Psoroptes cuniculi, Dermatophagoides farinae, Cheyletus malaccensis and Ornithonyssus bacoti, were collected and subjected to DNA barcoding. Sequences of cox1, 16S and 12S mtDNA, as well as ITS, 18S and 28S rDNA from mites were retrieved from GenBank and used as candidate genes. Sequence alignment and analysis identified 28S rDNA as the suitable target gene. Subsequently, universal primers of divergent domains were designed for molecular identification of 125 mite samples. Finally, the universality of the divergent domains with high identification efficiency was evaluated in Acari to screen DNA barcodes for mites. RESULTS: Domains D5 (67.65%), D6 (62.71%) and D8 (77.59%) of the 28S rRNA gene had a significantly higher sequencing success rate, compared to domains D2 (19.20%), D3 (20.00%) and D7 (15.12%). The successful divergent domains all matched the closely-related species in GenBank with an identity of 74-100% and a coverage rate of 92-100%. Phylogenetic analysis also supported this result. Moreover, the three divergent domains had their own advantages. D5 had the lowest intraspecies divergence (0-1.26%), D6 had the maximum barcoding gap (10.54%) and the shortest sequence length (192-241 bp), and D8 had the longest indels (241 bp). Further universality analysis showed that the primers of the three divergent domains were suitable for identification across 225 species of 40 families in Acari. CONCLUSIONS: This study confirmed that domains D5, D6 and D8 of 28S rDNA are universal DNA barcodes for molecular classification and identification of mites. 28S rDNA, as a powerful supplement for cox1 mtDNA 5'-end 648-bp fragment, recommended by the International Barcode of Life (IBOL), will provide great potential in molecular identification of mites in future studies because of its universality.

Study on the Relationship Between Microbial Composition and Living Environment in Important Medical Mites Based on Illumina MiSeq Sequencing Technology.

The microbiota of mites is closely related to their growth, development, and pathogenicity. Therefore, it is necessary to study the bacteria in mites. Here, for the first time, based on 16s rRNA V3-V4 region, the microbiota of 45 samples of nine species in six families of medically important mites were analyzed using Illumina MiSeq sequencing technique. The results showed that, at the phylum level, Proteobacteria (56.20-86.40%) were the dominant, followed by Firmicutes (6.41-19.43%), Bacteroidetes (5.56-13.38%) and Actinobacteria (1.93-28.07%). But at the genera the microbiota of mites are different, showing four characteristics: 1) The microbiota is related to the parasitic host. Demodex folliculorum (Acariforms: Demodicidae) and D. brevis (Acariforms: Demodicidae), both parasitizing humans, showed similar microbial composition, as did D. canis (Acariforms: Demodicidae) and Sarcoptes scabiei canis (Acariforms: Sarcoptidae) parasitizing dogs, but D. caprae (Acariforms: Demodicidae) parasitizing sheep showed unique microbial community; 2) The microbiota is related to mite's species. Dermatophagoides farinae and Cheyletus malaccensis (Acariforms: Cheyletidae), both collecting from flour, show respective microbial composition; 3) The microbiota is related to the life stage. There were differences in microbiota between adults and larvae of D. farinae, but no differences observed in Psoroptes cuniculi (Acariforms: Psoroptidae); and 4) The microbiota is related to the blood-feeding state. The microbiota of blood-fed Ornithonyssus bacoti (Parasitiformes: Macronyssidae) adults was significantly higher than that of unfed adults. This indicates that the microbiota of mites is affected by mite species, parasitic host, growth stage and habitat. Therefore, understanding these influencing factors will have a very important guiding significance for the prevention and control of mite-borne diseases.

Stress response and silencing verification of heat shock proteins in Dermatophagoides farinae under temperature stress.

Dermatophagoides farinae is a major exogenous allergen. Its ability to tolerate adverse external temperatures makes it responsible for widespread occurrence of allergies. Heat shock protein (HSP), a recognized temperature stress response gene, but its role in D. farinae remained unclear. Here, we performed a comprehensive study. First, we found that 25 °C was the optimal temperature, and all mites died at 48 or -20 °C for 1 h (LT100). Thus, 41 °C (LT15), 43 °C (LT25), 45 °C (LT45), and -10 °C (LT25) were selected as stress temperatures to perform de novo RNA-seq. Then, 17 main genes of the 47 differentially expressed HSP, were detected by qRT-PCR. Temperature and time gradient versus expression magnitude histogram revealed that HSP70, HSP83-1, HSP83-2, and HSP16-1 showed heat stress response only at 41-43 °C, while HSC71 and HSF played a regulatory role under both heat and cold stress, particularly HSF, with strong intensity, long duration, and quick upregulation at recovery for 10-20 min. Finally, gene expression and D. farinae survival rates significantly decreased following RNAi. These findings indicated that HSPs conferred thermo-tolerance or cold-tolerance to D. farinae. In conclusion, this was the first meaningful exploration that confirmed HSP and HSF playing an important role in temperature resistance of D. farinae.

De novo transcriptome sequencing and differential gene expression analysis of two parasitic human Demodex species.

Demodex are among the tiniest organisms in Acari and are important mammalian parasites. However, differences in pathogenicity between two human parasites, Demodex folliculorum and Demodex brevis, remain unknown. Related genetic studies are limited by RNA extraction difficulties and molecular data deficiencies. In this study, RNA extraction, de novo sequencing, functional annotation, and differential gene expression analyses were performed to compare D. folliculorum and D. brevis. This yielded 67.09 and 65.10 million clean reads, respectively, with similar annotations. Bioinformatics analyses and manual alignments identified 237 coding sequences comprising 48 genes from 29 families, including five important functional classes. Of these, 30 genes from 20 families related to metabolism, motion, detoxification and stress response, and allergic reaction were differentially expressed between the two species. Cathepsin type 1, serine protease inhibitor, arginine kinase, triosephosphate isomerase, muscle-specific protein 20-2, myosin alkaline light chain, troponin C, tropomyosin, and heat shock protein 90 were highly expressed in D. folliculorum, whereas cathepsin type 2, aspartic protease, serine protease, myosin heavy chain type 2, and alpha tubulin type 1C were highly expressed in D. brevis. Verified coding sequences were nearly consistent with unigene clusters. Further, absolute quantification results demonstrated that differentially expressed genes followed the predicted expression trend. Therefore, the first RNA sequencing and functional annotation analysis of two Demodex species was successful. Differential expression of important functional genes is likely implicated in pathogenicity disparities between these two species. Our study provides molecular data and technical support for further studies on human Demodex pathogenicity and functional genes.

Screening of reference genes and quantitative real-time PCR detection and verification in Dermatophagoides farinae under temperature stress.

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.

De Novo RNA-seq and Functional Annotation of Haemaphysalis longicornis.

PURPOSE: Haemaphysalis longicornis (Neumann) is a hematophagous tick widely distributed in northern China. It not only causes enormous economic loss to animal husbandry, but also as a vector and reservoir of various zoonotic pathogens, it spreads natural focal diseases, such as severe fever with thrombocytopenia syndrome, seriously threatening human health. Lack of transcriptomic and genomic data from H. longicornis limits the study of this important medical vector. METHODS: The engorged female H. longicornis from Gansu, China, was used for RNA extraction, de novo RNA-seq, functional annotation, and ORF prediction. RESULTS: As a result, 53.09 million clean reads (98.88%) with a GC content of 54.29% were obtained. A total of 65,916 Unigenes were assembled, of which 34.59% (23,330) were successfully annotated. Of these Unigenes, 22,587 (34.27%) were annotated to species by NCBI non-redundant protein (nr). Ixodes scapularis, Limulus polyphemus, Parasteatoda tepidariorum, Stegodyphus mimosarum, and Metaseiulus occidentalis were the top BLAST hit species, accounting for 47.23%, 9.58%, 4.11%, 3.50%, and 2.69%, respectively. A total of 29,182 ORFs were predicted, and 35 complete ORFs for functional genes were identified, including ORFs involved in digestion (14), stress responses (8), anticoagulation (3), reproduction (3), antimicrobial (2), drug resistance (2), movement (2), autophagy (1), and immunity (1), respectively. The Unigene ORFs encoding cathepsin and heat shock proteins were further analyzed phylogenetically. CONCLUSION: De novo RNA-seq and functional annotation of H. longicornis were successfully completed for the first time, providing a molecular data resource for further research on blood-sucking, pathogen transmission mechanisms, and effective prevention and control strategies.

Novel miR-1958 Promotes Mycobacterium tuberculosis Survival in RAW264.7 Cells by Inhibiting Autophagy Via Atg5.

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR- 1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.

LSU rDNA D5 region: the DNA barcode for molecular classification and identification of Demodex.

Whether ribosomal genes can be used as DNA barcodes for molecular identification of Demodex (Acariformes: Demodicidae) is unclear. To examine this, Demodex folliculorum, D. brevis, D. canis, and D. caprae were collected for DNA extraction, rDNA fragments amplification, sequencing, and analysis. The V2 and V4 regions of SSU rDNA; D5, D6, and D8 regions of LSU rDNA; and ITS region were obtained from the four morphospecies. BLAST analysis showed that the obtained sequences matched those of Demodex or Aplonobia (Acariformes: Tetranychidae) in Raphignathae. Phylogenetic trees derived from V2, V4, D5, D6, and D8 regions, but not from ITS region, showed that the four species of Demodex clustered independently. Sequence divergence analysis further demonstrated that D5, D6, and D8 regions had obvious barcoding gap between intraspecific and interspecific divergences, with the gap of D5 (16.91%) larger than that of D6 (11.82%) and D8 (4.66%). The V2 and V4 regions did not have a barcoding gap, as the intraspecific and interspecific divergences partially overlapped. For the ITS region, intraspecific and interspecific divergences completely overlapped. These results suggest that the D5, D6, and D8 regions of LSU rDNA, especially D5, are suitable DNA barcodes for Demodex.

Function of heat shock protein 70 in the thermal stress response of Dermatophagoides farinae and establishment of an RNA interference method.

Dermatophagoides farinae are an important mite species that cause stored product deterioration and allergic diseases. They widely breed in human habitats because of their strong tolerance to extreme external temperatures. However, mechanisms underlying the stress response and tolerance of D. farinae are unclear. We hypothesized that heat shock protein 70 plays an important role in the heat stress response of D. farinae. In this study, we determined the survival rates of D. farinae at high temperatures (37 °C-45 °C) by performing temperature-gradient experiments in vitro and assessed the expression level of HSP70 by performing RT-qPCR. First, we confirmed that HSP70 regulated the heat stress response of D. farinae, with maximum heat stress regulation observed at 41 °C. Next, we confirmed the presence of a Dicer enzyme-mediated RNA interference (RNAi) pathway in D. farinae by searching the NCBI database and a Dicer site prediction website. Finally, we performed RNAi in D. farinae by using an immersion method with screened dsHSP70 fragments. Moreover, we performed concentration-gradient experiments to determine that 600 ng/µl was the minimal effective concentration of dsHSP70 for silencing HSP70. These results confirm that HSP70 regulates the heat stress response of D. farinae. The present study is the first to report the use of the non-invasive and highly sensitive immersion method for performing RNAi in D. farinae. The results of the present study provide a technical foundation for performing functional gene research and for developing molecular prevention and control strategies against medically important mites.

The Construction of Full-Length cDNA Library for Otodectes cynotis.

BACKGROUND: Otodectes cynotis (Hering, 1838) is the pathogen of otodectic mange distributed worldwide. The mite mainly infests carnivores and, sometimes, humans. However, due to the lack of cDNA library, research on its pathogenesis has been challenging. METHODS: To solve this problem, the present study first sampled O. cynotis mites from an infested cat from Xi'an, China, for RNA extraction. Then, the full-length cDNA library was constructed using the SMART technique. Finally, positive clones > 500 bp and Hsc70-5 gene fragment specifically amplified from the cDNA library were sequenced and analyzed to verify the library's reliability. RESULTS: Results showed that RNA extracted from 300 mites had good quality with a concentration of 149 ng/µl and OD260/OD280 of 1.99. The library satisfied the quality standard of a good library with a titer of 5.02 × 105 PFU/ml and a combination rate of 97.61%. In addition, clone 4 and Hsc70-5 showed 98.38% and 99.72% identity with Ef1-α and Hsc70-5 gene sequences of O. cynotis in GenBank, respectively. CONCLUSION: The cDNA library of O. cynotis constructed here was successful and reliable, creating the basis for research on RNA sequencing and functional genes of O. cynotis.